CA2330172A1 - Prosthetic implant and methods of use for therapeutic gene expression - Google Patents

Abstract

The invention provides a method of systemic delivery of a gene product to an individual. The method consists of implanting within the wall of the digestive tract of en individual a prosthetic chamber containing an effective amount of cells secreting the gene product.
The prosthetic chamber can be implanted within or proximal to a connective tissue layer including, for example, between the tunica across and the tunica muscularis of the digestive tract wall. The gene product to be delivered can be selected from cytokines, hormones, growth factors and blood clotting factors including, for example. Epo or insulin.
Expression of the gene product can be constitutive or inducible.
Therefor, the invention provides a method of treating a pathology mediated by the deficiency of a gene product. The method consists of implanting a prosthetic chamber containing an effective amount of cells expressing therapeutic levels of the deficient gene product.

Description

2 PCf/US99/1Z116 This invention was made with government support under grant numbers DK 43727, DK 47754 and DK 506H6 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.
This invention relates to generally to methods of gene delivery and, more specifically to cell implantation methods for sustained engraftment and long term gene expression.
The availability of recombinant cloning and expression methods has provided significant advances in the therapeutic treatment c7f human diseases. For example, the availability of recombinant human erythropoietin (Epo), a regulator of red blood cell production and maintenance, has provided a significant. advance ire the treatment of renal failure patients receiving dialysis with the elimination of attendant dangers of transfusion therapy and an increase in the quality of life of these patients. The administration of recombinant Epo is now widely used for long-term treatment of anemia associated with chronic renal failure, cancer chemotherapy, and human i..mmunodeficiency virus infections. Long-term, sustained delivery of this hormone, and others like it, by cell therapy :rather than by repeated injections would provide substantial. clinical and economic benefits. Unfortunately t:he DNA regulatory sequences which control the expression of Epo in response to tissue oxygenation are toa large to be suitable for insertion in a retro viral vector. Similar problems exist Lor the expression of other hormones and therapeutic proteins as well.
Long-term in vivo gene expression requires both target cells and gene delivery vectors that permit continuous vector encoded activity. of the three prevalent virus-based methods of gene transfer, retro viral vectors are likely the most useful for ex vivo gene transfer.
Adeno-associated virus(AAV) vectors have some attractive features, such as safety and abil.i.ty to transduce non-proliferating ce:Lls brat do not possess advantages over retro viruses for ex-vivo gene transduc~ion. Replication defective retro viral vectors can be made with high titers, will infect a wide variety of cell. types and infection results in stable proviral integration into the host chromosome providing gene expression for the lifetime of the cell and its progeny. Therapeutic genes can be expressed at high level from the viral long terminal repeat (LTR) promoter/enhancer o r strong internal promoters and the incorporation of internal ribosome entry sites from picornaviruses into retro viral vectors has allowed the generation of bicistronic vectors conferring linked-gene selection. However, there is always great concern when using replication defective vectors because contamination with a single replication compentant vector can result in infection of the patient with a disease producing virus.
Non-hematopoietic cells studied as alternative vehicles for gene therapy include skin fibroblasts, myoblasts and vascular smooth muscle cells. Skin fibroblasts are easily obtained, cultured and transduced but have a major disadvantage oi~ inactivating vector sequences after transplantation. Myoblasts represent one target cell type for gene therapy. Transduced skeletal myoblasts have been uaed to de:L.i ver Epo in mice and to wo m6issz pcrrtrs~nziiu treat alpha-L-iduronidase deficiency in dogs following transplantation. Additionally, intramuscular injection of plasmid DNA has produced systemic expression of Epo and other therapeutic proteins in mice.
Smooth muscle cells are present within the vasculature as a multilayered mass of long-lived cells in proximity to the circulation and also have been investigated as targets for gene therapy. Moreover, transduced vascular smooth muscle cells seeded into carotid arteries in a rat model has provide sustained expression of both marker and therapeutic genes, including for example Epo and granulocyte-colony stimulating factor (C-CSF).
However, this procedure is significantly hindered far human therapeutic applications because it requires arterial injury to achieve cell engraftment.
Thus, there exists a need for a reliable and reproducible method which provides long-term sustained gene expression of a therapeutic or beneficial protein with minimal adverse side effects. The present invention satisfies this need and provides related advantages as well.
The invention provides a method of systemic delivery of a gene product t.o an individual. The method consists of implanting within the wall of the digestive tract of an individual a prosthetic chamber containing an effective amount of cells secreting the gene product.
The prosthetic chamber ran be implanted within or proximal to a connective tissue layer including, for example, between the tunics serosa and the tunics muscularis of the digestive tract wall. The gene product to be delivered can be selected from cytokines, hormones, growth factors and bland clotting factors including, for example, Epo or insulin. Expression of the gene product can be constitutive or inducible. Therefore, the invention provides a method of treating a pathology mediated by the deficiency of a gene product. The method consists of implanting a prosthetic chamber containing an effective amount of cells expressing therapeutic levels of the deficient gene product.
~IRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a schemati~~ cross section of a implantation site under t:he tunica serosa of the stomach.
Figure 2 shows the hematocrit values of animals implanted with Epo-secreting, LrEpSN-transduced (closed symbols), and ADA-scret:ing, hASN-transduced (open symbols , vascular smooth muscle cells as a function of time.
Figure 3 shows histological cross-sections of PTFE chambers containing transduced smooth muscle cells implanted in the stomach.
Figure 9 shows expression levels of endogenous Epo mRNA in animals rece:i.ving Epo-secreting cell implants compared to ADA-secreting control cell implants.

wo 99isusz Pc rmss9nzm This invention is directed to cell transplantation methods for the long-term, sustained delivery of a therapeutic or beneficial protein product.

5 The methods of the invention employ a biocompatable prosthetic chamber surgically implanted into an individual and seeded with the cells expressing a therapeutic or beneficial gene product. The implanted cells become engrafted, providing consistent, long-term expression of the therapeutic product. The method is safe, enables easy engraftment and removal of implanted smooth muscle cells, and can be applied generally to the systemic delivery of a variety of therapeutic proteins including, for example, cytakines, hormones, enzymes and clotting factors.
In one embodiment, the prosthetic chamber is implanted at the tissue plane between the tunica serosa and the tunica muscularis which provides a rich growth environment for retention of transduced cells.
Therefore, one advantage of the method is that this tissue is composed of smooth muscle cells, is well vascularized and able to provide nutrition for implanted cells with concommitant secretion of the gene product into the circulatory system.
In another embodiment, the methods of the invention implant under the tunica serosa genetically modified vascular smooth muscle cells for stable long-term cell engraftment expression of Epo. Smooth muscle cells are present at this location and advantageously potentiate the engraftment efficiency and long-term survival of the transplanted cells because they are a normal constituent of the implanted area. In this embodiment, autologous transduced cells are injected into a chamber created from a polytetrafluaroethylene (PTFE) ring placed between the serosa and muscularis of the stomach. The engrafted PTFE ring becomes vascularized and permits both the long-term survival of transduced smooth muscle cells and an area protected from ingrowth of non-transduced cells.
As used herein, the term "systemic" when used in reference to expressian of a gene product is intended to mean delivery of the gene product to the circulatory system of an individual. The circulatary system includes those parts of the body receiving blood through either the aorta or the pulmonary artery.
As used herein, the term "delivery" is intended to mean presenting to the hematopoietic system the expressed gene product.. Presentation ~~an include, for example, direct secreta.on into the circulatory system or indirect secretion into the extracellular space such that diffusion into capillary beds occurs. Presentation also can include surface expression an the plasma membrane of the implanted cells where cells oz t:he hematopoieti.c system can come in contact with the expressed gene product. Specific examples of ser_reted gene products include direct or indirect secretion of cytokines, hormones, growth factors, extracellular matrix proteins and vaccine antigens. Delivery of cell. surface proteins can include, for example:, cell vaccines which express tumor antigens. Those skilled in the art will know which mode of delivery is applicable to achieve a particular result for a particular gene product.
As used herein, the term "gene product" is intended to mean both polypeptide and nucleic acid WO 99/62562 PC'T/US99112116 products that can be expressed or replicated from a nucleic acid. Specifi~~ examples of a polypeptide gene product includes peptides, polypeptides or proteins encoded by structural genes. Specific examples of a nucleic acid gene product includes RNA such as mRNA, antisense RNA or DNA and viral vectors for example.
As used herein, the term "'implanting" is intended to mean the introduction or transplantation of cells in a prosthetic ~~hamber into an individual wherein the cells remain viable after implantation and maintain their gene product expression capability. Implanting includes, for example, directly introducing the cell-containing prosthetic ~~hamber into a tissue, introduction of the cell-containing prosthetic chamber with accessory cells which are capable of grafting to the surrounding tissue or which facilate long-term stability of the prosthetic chamber or gene product expression capability of the implanted cells, Such accessory cells can be, for example, Located within the prosthetic chamber, attached to, or, associated with the perimeter of the chamber.
Implanting additionally includes, for example, introduction of the cell-containing prasthetic chamber into the tissue with other components such as extracellular matrix components, fragments or other molecules which facilitate adhesion of the cells as well as with soluble or substrate bound growth factors, for example. Implanting is generally performed by surgical procedures but other methods can similarly be employed so long as the manipulation is compatible for use with the particular type of prosthetic chamber. being used in to implant the cells. Those s killed in the art will know, or can determine, which mode of introduction is appropriate for a particular type of chamber.

As used herein, the term "prosthetic chamber"
is intended to mean a devise that is capable of compartmentalizing cells. Compartmentalization allows the initial separation of implanted cells from endogenous cells within the implanted area without substantially affecting the exchange of nutrients,. cell by-products and factors necessary for cell viability and proliferation.
The compartmentalization effects performed by the prosthetic chamber should also allow the cells to maintain their gene product expression capability.
Barriers which form the compartment can be temporary or permanent and can effect the complet=a encasement of the cells, or alternatively, can allow growth of the implanted cells over and around the barriers as the cells become engrafted and enlarged in the area of implantation. Temporary barriers can include, for example, biodegradable material whereas permanent barriers can include, far example, metals such as titanium and stainless stee.l., synthetic polymers and biopolymer. The sizes of such protheti.c chambers can vary depending on the number. of cells t:o be transplanted and can be about lOmm or greater, between about 7-l.Omm and preferably between about 3-6mm in outer diameter or width. However, sizes larger or smaller can be used which can accommodate a desired number of cells or which is suitable for a particular locatian for implantation.
Those skilled in the art will know what sizes within the above range of sizes, as well as prosthetic chambers that lager or smaller than these sizes depending on the intended application.
Compartmentalization using partial physical barriers that contain the cells during implantation but are open to the surrounding cells and tissue components in one or more places includes, for example, devices g having shapes and structures in the form of a ring, cylinder, horseshoe, planer porous structures or any variation thereof which functionally results in containment of the cells at implantation. A specific example of a prosthetic chamber that compartmentalizes cells using partial physical barriers is a ring of polytetrafluoroethylene (PTFE). Compartmentalization can also be accomplished thraugh the use of a three-dimensional biomatrix containing the cells to be implanted, beads or particles coated with cells being attached through receptor ligand interactions as well as any combination of such compartmentalization devices and biomatricies. Examples of biomatxix include extracellular matrix, including for example, collagen gels. An example of a combination of compartmentalization de~ri.ses includes cells casted in a collagen gel contained within a PTFE ring.
Complete encasement of cells includes, for example, the use prosthetic chambers constructed of semipermeable materials, such as membranes, that are barriers to cells but allow passage out of the barrier of nutrients, cell by-products, growth factors and secreted gene products. For example, cells can be placed into an ampule or sack constructed of this material and then physically sealed. The semipermeable barrier also can be coated with a biomatrix such as ext.racellular matrix, or the cells can be contained in a biogel matrix or attached to particals coated with extracellul.ar matrix components or cell attachment fragments thereof, for example.
As used herein, the term "effective amount"
when used in reference to the number of cells secreting a gene product is intended to mean the number of cells that can be implanted and become engrafted so as to secrete a WO 00147754 ~0 PCTlEP00/01524 Inhibiting the expression of a target gene in the genome of a plant may be desired in many purposes. Many transcription factors known by the man skilled in the art play a role in the control of metabolic pathways (starch, lipids, amino acids...) or are involved in the plant development, or in the plant s sensibility to pathogen. For example, blocking a gene whose expression is necessary for pollen or another formation (e.g. the Ms-41-A transcription factor, as above described) produces male sterility. Blocking the gene controlled by the AP-3 transcription factor also leads to male sterility. As another example, blocking the gene which codes for the enzyme which catalyses the conversion ~o of sugars to starch can be used to produced sweet corn (see EP 475 584).
It is also possible to obtain transgenic plants with enriched content in lysine by using, according to the invention, the opaque 2 transcription factor (Schmidt et al., 1990), involved in the control of the expression of certain zeins. One could further use Myb-related transcription ~s factors involved in the control of anthocyanin biosynthesis in flowers (Martin et al., 1991 ; Matin, 1997), to madify their colour. Use of other members of Myb-related transcription factors playing a role in the regulation of phenylpropanoid and lignin biosynthesis (Tamagnone et al., 1998) could also be interesting.
Some others could be involved in cellular development and senescence.
The chimeric construct, fusion of the repressor domain to the plant transcription factor or part of it, can be advantageously used to identify essential protein-protein interaction domains and interacting protein partners in planta in transgenic plants.
2s In a preferred embodiment, expression of a chimeric protein comprising the repressor domain in fusion to a plant transcription factor is known to cause a dominant phenocopy. By the deletion of increasing parts of plant sequences encoding the transcription factor and their repression in fusion to the repressor domain in transgenic plants, essential protein domains can be 3o identified. These are DNA-binding domains but also others, like protein-protein interaction domains, if the transcription factor is part of multi-component complex. The method according to the invention is therefore also suitable to study protein-protein interactions in planta.

implanted in an individual can provide therapeutic effects in vivo.
The invention provides a method of systemic delivery of a gene product. The method consists of implanting within the wall of the digestive tract of an individual a prosthetic chamber containing an effective amount of cells secreting said gene product.
The methods of the invention employ cell engraftment as a vehicle for the systemic delivery of a gene product. Long-term engraftment ol: cells expressing a desired gene product is facilitated through the use of a prosthetic chamber. The chamber :is surgically implanted at the site of engraftment and cells are seeded into the chamber either during or previous to implantation. The function of the prosthetic chamber is to contain cells at the site of implantation, which provides an initial favorable microenvironment for cell viability with an increase the engraftment efficiency of implanted cells.
The location of the prosthetic chamber can be essentially any tissue an an individual that is accessible to surgical procedures and will depend on, for example, the type of prosthetic chamber used, the cell type being implanted and the gene product or expression elements employed for production of the gene product.
Those skilled in the art will know, or can determine a suitable location for implantation given the teachings and guidance provided herein. Generally, a suitable location for implantation of the prosthetic chamber is a tissue, or region of a tissue which is adeguate:Ly vascualrized and capable of supplyi:~g nutrients and growth factors necessary for' cell survival. Such tissues WO 99/62562 PGT/US99I1=116 can include, for example, muscle including skeletal and smooth muscle, connective tissue, epithielia and endothelia cell linings, basement membrane structures, epidermis and dermis.
Certain tissues and organs have morphologies that provide additional advantages for the use of prosthetic chambers as described herein because they naturally form a stable compartment which further facilitates the function of the chamber. Implantation into these regions allows for the use of a broad range of prosthetic chamber types. 'tissues which exhibit favorable morphologies include essentially any tissue in which there is a plane between two :Layers of tissue which are vascularized and can stably support the surgical insertion of a prosthetic chamber. For example, the layers of the digestive tract is one such tissue lacation that exhibits a morphology favorable for implantation.
Briefly, the wall of the= digestive tract is composed of four layers. The first, outermost :layer is a thin layer of mesothelium and connective tissue and is known as the tunica serosa or seros<~,. The next layer inward is the tunics muscularis, composed of muscle layers, which is followed by a second 1<~yer of connective tissue known as the submucosa. The innermost layer also contains connective tissue and :is additionally lined with a layer of epithelial cells. rchis inner layer of the digestive tract is known as the mucosa. All cyf these layers are well. vascularized which wi.l.l support. cell growth and viability. Therefore, a prosthetic chamber can be implantated into, or be=_tween any of the above .layers and seeded with cells secreting a desired gene product to achieve systemic delivery of the product.

WO 99/62562 PCTNS99/1=11b A prosthetic chamber can be implantated into any of the above described tissues or morphological tissue layers to form a microcompartment for cells secreting a desired gene product. Insertion at a plane between tissue layers :is one location which can be used for the efficient implantation of a prosthetic chamber.
For example, the plane between any of the four digestive tract layers described above are easily accessable by routine surgical procedures, can be separated without substantial damage to the morphological structure or biological function of the tissue layer and inherenty provide a natural crevice once segregated for which a prosthetic device can tie stably placed and seeded with cells. For example, the muscle cel'~. bed at the plane between the tunics muscularis and the tunics serosa on the stomach can serve as a location to receive a prosthetic chamber occupied by cells expressing a gene product of interest. This tissue site is well vascularized and will therefore provide nutrition for implanted cells. The long-term survival of implanted cells requires access to nutrients such as oxygen from the blood supply. As discussed further below, smooth muscle cells are a normal part of the architecture of this site. Modifying this cell type to secrete the desired gene would therefore provide additional advantages for the survival of transplanted cells because they are a normal constituent of the targeted area. A
specific example of impimplanting vascular smooth muscle cells retrovirally transduced to express erythropoietin and G-CSF is described further below in the Examples.
Implantation of a prosthetic chamber can be accomplished by surgical placement cf the chamber into any of the tissues or morphological tissue layers described above. Briefly, the chamber is implanted by WO 99/62562 PGT/US99l1Z116 making an incisian at the entry site of the appropriate depth and length to expose she target tissue and physically placing the chamber within the tissue incision. Alternatively, the target tissue can be externalized, followed by one or more incisions until an area in the target location has been created for implantation. Methods for implanting a wide variety of artificial devices and prothesis as well. as cell, tissue and organ transplantation and engraftment procedures are well known to those skilled in the art. Similarly, a wide variety of surgical methods applicable for the treatment of diseases, diagnostic indications and cosmetic procedures are also well known to those skilled in the art and are all applicable surgical methods which can be used for implanting a prosthetic chamber of the invention. For example, the use of endoscopes is well known to those skilled in the art and affords a minimally invasive procedure to access and perform surgery on the stomach and intestin. ~larious modification of and combinations of such mei~hods known to those skilled in the art similarly applicable for implanting one or more prosthetic chambers in an individua.L far the systemic delivery of a gene product. Specif:LC examples of a surgical procedure which can be used far implanting a prosthetic device and seeding with cells modified to express a desired gene product is described further below in the Examples.
Therefore, the invention provides a method of systemic delivery of a gene product where a prosthetic chamber containing an effective amount of cells secreting said gene product is implanted within ar proximal to a connective tissue layer of the digestive tract of an individual. The location cao be, for example, between the tunica serosa and the tunica musclaris of the digestive tract wall.
The invention also provides a method of systemic delivery of a gene product by implanting a 5 prosthetic chamber containing an effective amount of cells secreting said gene product where the prosthetic chamber has an outer diameter or width that is greater than about lOmm, preferably between about 7.0-lOmm, more preferably between about. 3-6mm. The prosthetic chamber 10 can consist of various shapes and can be open at one or more locations to the surrounding tissue, or it can be completely enclosed.
Prosthetic chambers used in the methods of the invention can be of various shapes and sizes so long as 15 they function to restrain cells seeded into the chamber at the time of implantation and can be surgically manipulated into a desired location within the recipient individual. As described previously, the chamber can either partially or completely encase t:he cells to provide a beneficial microenvironment for cell growth, viability and long-term engraftment. Prosthetic chambers which particially encase seeded cells include chambers open at one or more locations to the surrounding tissue.
Such chambers exhibit the advantage in that they allow for expansion of the engrafted ells by proliferation and migration out of the chamber into the surrounding tissue regions.
Shapes of such prosthetic chambers include, for example, a ring, horseshoe and cylinder or any geometric shape or motif that contains an inner region which can contain seeded cells and an outer barrier which serves to restrain the cells. For example, a ring having an inner diameter of about 4mm for seeding cells and an outer diameter of about 6mm is one example of a prosthetic chamber that is partially open to surrounding tissue and capable of containing cells at the site of implantation.
Cells seeded into the center of the ring are initially restrained by the outer barrier at implantation. As the implanted cells engraft and expand they are able to migrate out of the ring and over the outer barrier forming a patch of stably engrafted cells. Other shapes having similar functional features such as those listed above as well as squares, hexagons, rectangles, triangles and the like also can be used for a prosthetic chamber in the methods of the invention. Therefore, the shape of the chamber is unimportant so long as the chamber 25 functions to contain cells seeded at the site of implantation.
Prosthetic chambers having various alternative forms to those described above also can be used in the methods of the invention. For example, prosthetic 2o chambers composed of a planar, porous material will similarly function to contain cells at the site of implantation, allow access to the surrounding tissue and exchange of nutrients, factors and cell by-products.
Cells can be seeded into the pores which completely, or 25 partially extend, through the planar material. One advantage with this structure for a prosthetic chamber is that the surface for cell containmE~nt is increased portional to the number and pores. Additionally, cells can be seeded onto the outer so rfa<:es of the plane by, 3o for example, coating it with cell adhesion proteins yr substrates and allowing the cells to ;attach through receptor-ligand interactions. Concave disks of various shapes and depths can similarly furocr.ion as a prosthetic chamber to contain cells at the sit::e of implantation. As wo m6zs6i Pcrms~nzn6 with porous chambers, concave disks can be coated with adherent proteins to facilitate cell attachment at the time of implantation or, alternatively, preseeded with cells prior to implantation.
Prosthetic chambers that can be used in the methods of the invention also can be of various three-dimensional shapes and sizes. For example, the chambers can be cylinders and cones which have an inner area for containment of the cells and an outer barrier.
Additionally, chambers can be particles or spheres which can be coated which adherent cells ;prior to or during implantation. The particles or spheres can be porous or textured so as to increase the surface area and therefore the number of cells r_apable of being implanted using such a chamber. Therefore, the chambers which can be used in the methods of the invention are not limited to a particular geometric shape, structure or three-dimensional form.
The selection of a particular shape or form for use in the methods of the invention will depend, for example, on the site of implantation, number cells to be seeded and surgical preference by those skilled in the art. For example, if the prosthetic chamber is to be implanted between tissue layers, such as between the tunica serosa and the tunica muscularis of the digestive tract, those skilled in the art can select, for example, a ring, horseshoe, other geometric shape, or other planar chamber that can be inserted between the tissue layers with minimal damage and preturbat.i.on of the surrounding tissue. Those skilled in the art. will know, or can determine, what shape and structure of the prosthetic chamber is appropriate fear a particular tissue location.

WO 99/62562 PCT/tJS99/1Z116 Prosthetic ctuambers which completely encase seeded cells include chambers such as spheres, ampules, and sacks constructed ~:of semipermiable barriers. Such chambers can be advant~:~geous when using non-autologous cells because they physically block host immune cell attack and therefore reduce graft rejection by this mechanism. The use of such completely enclosed prosthetic chambers therefore allows for the implantation of non-autologous cells and reduces or eliminates the need for immunosuppressa.ve drugs which compromise the recipient's immune sysr_em. The shape of such chambers can additionally be any of those described previously except that the exposec:~ areas of the previously described chamber shapes would instead be enclosed with a semipermeable barrier. for example, a ring can be used where the top and bottom planes are enclosed with, for example, a semipermeab:Le membrane, thus forming an enclosed cylinder or oval-shaped structure having cells seeded into the internal area prior to implantation.
The prosthetic: chambers used in the methods of the invention can be of various sizes and will depend on the desired number of :ells to be implanted, in addition to the site of implant<~tion and associated considerations described above in reg<srd to selection of the shape of the chamber. For example, a ring having an inner diameter of about 9mm will accommodate about 1x:.0 or greater number of cells. Larger chambers larger will accommodate proportionally more cells and conversely, smaller chambers can bs~ used to implant proportionally less cells. In additi<:~n, the total number o:f cells car.
be substantially increased by about 2- to 10-fold, or more if porous or text'.ired chambers are used. The chambers therefore can k}e essentially any size which the tissue at the site of implantation c~an physically WO 99/6iS62 PCTNS99/12116 accommodate and which is in acceptable medical parameters for implantation of a prosthetic device.
For example, a prosthetic chamber between about 7 and lOmm or greater in outer diameter or width and as small as 3 to 6mm can be accomodiated at the tissue plane between the tunica serosa and the tunica muscularis. The inner diameter or width of a prosthetic: chamber used in the methods of the invention similarly can be within these same size ranges and can depend, for example, an the type of material used and the methods of fabrication.
The height of the prosthetic chamber is generally about 3mm to allow sufficient diffusion of oxygen into the implanted cell area. However, the height of a chamber can be as small as 1-2mm and as large as 9-5mm or greater. Moreover, the height can be of various other sizes depending on the site of implantation and the shape and surface area exposed to the surrounding tissue and vasulature. Those skilled in the art will know how to vary the height of the prosthetic chamber to achieve sufficient diffusion of axygen and other nutrients to maintain viability and support engraftment of the implanted cells given the teachings and guidance provided herein.
The above described prosthetic chamber sizes will accommodate populations of cells from about 1x10' or smaller. The size of the cell population to be implanted can be, for example, as small as 1x10' or less. However, larger populations such as 1x10', 1x10, 1x10' and ,yxl0~
are, in general, the amount of implanted cells that will secrete a sufficient amount of the desired gene product to accomplish the targeted functional effect. Therefore, any of the prosthetic chambers described above, as well zo as others known to those skilled in the art. are applicable for use in the methods of. the invention The number of cells required for an effective amount will vary depending on the gene product being secreted, its expression and secretion level into the extracellular space arid its inherent biochemical activity. For example, a gene product that has a high affinity for substrate or ligand will inherently require less amounts to be secreted to achieve the same level of l0 activity when compared to a lower affinity homologue.
Similarly, a gene product with a longer half life also will require less amounts or rate of secretion to achieve comparable activities or accumulation levels to a shorter lived gene product. Therefore, an effective amount of cells will vary depending on inherent properties of the gene product to be secreted and expression characteristics of the modified cells, all of which are criteria well know to those skilled in the art.
Therefore, the invention also provides a method of delivering a gene product systemically by implanting an effective amount of cells secreting the gene product where the number of cells is a population of about 1x20' cells or greater, preferably about 1x10' cells or greater, more preferably about 1x10' cells or greater.
Fabrication materials for the prosthetic chambers can be of any solid, porous or degradable material so long as it is biocompatable. For example, the chamber composition can include surgical grade metals, synthetic polymers and b~~.opolymers. Examples of synthetic fabrication material is polytetrafluoroethylene (PTFE), gortexG~ and carbon fibe r Surgical grade metals include, for example, titanium and stainless steel WO 99/6?.562 PCf/US99/1I116 whereas bivpolymers can included, for example, collagen, fibronectin, proteoglycans and active fragments thereof.
The prosthetic chambers can additionally include a wide variety of intracellular and systemic biomolecules that augment cell viability and proliferation of implanted cells. Such biomolecules can include, for example, growth factors and hormones. Additionally, prosthetic chambers containing semi-permeable barriers can be composed of, for example, natural or synthetic membranes with a pore size that excludes cell-cell contact.
Generally, a pore size of about 0.22 mm is sufficient to allow exchange of macromolecules such as polypeptides and proteins. However, other pore sizes can also be used without affecting the function of the chamber ar of the implanted cells. Specific examples of chambers made from some of these materials are described below and in the Examples. Other materials known to those skilled in the art also can be used for the prosthetic chambers used in the methods of the invention.
Briefly, prosthetic chambers used in the methods of the invention can be fabricated with PTFE.
This material is non-immunogenic, easily fabricated into various shapes, sizes and formats, can be prepared for implantation using methods well known to those skilled in the art for sterilization arid handling and can be manipulated by routinely surgical procedures for implantation. Prosthetic chambers also can be fabricated using various lengths of 18g cerclage wire, or surgical grade stainless steel wire, which is bent into a "U" or horseshoe shape to restrain cells seeded into the chamber. Modification to the prosthetic chambers can additionally be made t:o help maintain the shape and volume of the chamber. For example, short lengths or disks of about 0.5-lcm in length or diameter and about lmm thick PTFE or other compatible material can be inserted in the chamber after the cells are introduced to achieve this effect. A fur~.her modification includes casting the cells that are to be grafted into the chamber in a contracting collagen matrix which serves to both concentrate the cells and to hold the cells in place after engraftment. This type of prosthetic chamber is applicable with any bi.ocompatible material that can be manipulated into a shape to contain cells. The inner diameters of any of such chambers can exceed 10 mm and allows for the engraftment of at least about 1x10' or more modified cells.
Systemic delivery of a desired gene product utilizes cells as a vehicle for delivery and long-term production of the gene product in the recipient individual. The cells can be modified ex vivo to express in a secreted form the desired gene product, or alternatively, the imply nted cells can be natural producers of the gene product. Cell. implantation and engraftment is facilitated using the prosthetic chambers as described above. The cell type t:o he implanted can be any cell type, population of cell types and heterogeneous mixtures, that ar.e compatible with the site of implantation and the recipient's major. histocompat:ibility (MHC) antigens responsible for graft rejection.
Compatibility includes modification of the implanted cells or treatment of the recipient so as to allow self-recognition by the recipient's immune system or to down regulate the recipient'; immune system by immunosuppressive agents. Therefore, and as described further below, selection of the cell type to be implanted follows criteria for ce.l.l., tissue and organ engraf~ment and/or transplantation which is well known to those skilled in the art.

WO 99162562 PGT/US99llZlll Cell types to be selected for generating the modified cells used in the methods of the invention are those which are capable of polypeptide synthesis and secretion and compatible with the recipient's immune system and the site of implantation. With the exception of highly specialized cell types, such as red blood cells, the large majority of cells meet these criteria and can be used for constructing the modified cells used in the methods of invention. Alternatively, cells which naturally express the desired gene product can be obtained, for example, from a donor individual or ;.issue and implanted directly without the need for genetic modification.
The cell type chosen for .modification is selected according to the biological characteristics of the cell and according to gene expression criteria well known in the art. For example, abjective criteria such as the ease of culture efficiency, the ease of genetic modification and other routine cellular and molecular manipulations can be used to evaluate and select the cell type for modification. Those cell types which can be passaged in culture for multiple generations without substantial loss in viability are preferable candidates for modification as systemic delivery vehicles for a desired gene product. As will be described further below, such cell types include, for example, both primary cells as well as cell lines. Additionally, criteria such as the proliferation characteristics can also be evaluated for selection of the cell type to be modified.
Cell types are additionally selected according the efficiency with which they can be modified to express and secrete a gene product in the methods of the invention. Cell types that can be readily modified and WO 9916Z'S62 PCTNS99I1Z116 selected for the expression of the introduced gene or genes by any of a variety of methods known in the art are applicable for constructing the implantable cells of the invention. Availability of promoter and regulatory elements can also be included as a criteria for selecting a particular cell type for modification. Such characteristics and criteria are routine and well know to those skilled in the a.r1-_.
Various combinations of the above exemplary characteristics as well .~s other characteristics can additionally be used far selecting ,~ cell type to modify.
For example, if the objective is to achieve a particular level of gene praduct, sc~c:retion using a relatively small number of cells, then a cell type that can be efficiently modified to yield high levels of expression can be selected to achieve the desired result. In contrast, if cell number is nat a limiting factor, then it can be desirable to base selection of the cell type on favorable growth or proliferation characteristics. Additionally, various expression elements can be utilized to augment or modulate the level of expression. and secretion so as to complement advantageous characteristics or to overcome any deficiencies of the chosen cell type for modification. Such criteria and characteristics are well known or can be determined by those skilled in the art.
The cells selected for use in the methods of the invention can therefore originate from essentially any tissue or organ. However, the engraftment efficiency can be inherently augmented by selecting a cell type or composition of cell types that are natural constituants of the tissue where the prosthetic chamber is to be implanted. For example, cellular constituents of the layers of the digestive tract Include connective tissue of mesothelial origin, muscle and epithelial cells.
Therefore, selecting one of these cell types for implantation at the corresponding tissue layer will yield engraftment into an environment of similar or identical 5 cells and provide a compatible habitat for cell. viability and growth. A specific example, is the implantation of modified smooth muscle cells between the layers of the tunica serosa and the tunica muscularis of the digestive tract because the latter. layer is composed primarily of 10 smooth muscle cells.
Another specific example of a cell type that is useful for engraftment with the methods and prosthetic chambers of the invention is a fibroblast cell.
Fibroblast cells can be obtained from a variety of tissue 15 sources including, for example, skin, liver, muscle or arterial tissue. Fibroblast cells are also advantageous in that they are easily obtained and isolated, routinely modified, and can proliferate to higher densities within a graft. For example, a 10 cm= area within a prosthetic 20 chamber can contain 10' fibroblast cells, or up to 20 layers of cells, and can secrete essentially any desired gene product at effective amounts to achieve therapeutically beneficial results.
Therefore, the invention provides a method of 25 systemic delivery of a gene product where cells secreting the gene product are smooth muscle cells or fi.broblasts.
The gene product can be constitutively secreted, or alternatively, it can be secteted by i.nducible regulation.
For implanting primary cells, a tissue and therefore a site for implanting a prosthetic chamber, should be selected that is easily accessible arzd contains WO 99/6Z56Z PCTlUS99/IZI I6 cells that exhibit desirable growth and expression characteristics such as those described herein.
Additional considerations when selecting a tissue source include choice of a tissue that contains cells that can be isolated, cultured and modified to secrete a desired gene product. In addition to the cellular constituents of the digestive tract layers, other examples of cell types that can be modified t.o secrete a desired gene product include, for example, muscle (smooth, skeletal or cardiac), fibroblast, liver, fat, hematopoietic, epithelial, endothelial, endocrine, exocrine, kidney, bladder, spleen, stem and germ cells. Other cell types are similarly known in the art that are capable of being modified to secrete gene products and can similarly be obtained or isolated from a tissue source from a recipient or donor individual. Although human tissue sources are advantageous for therapeutic purposes, the species of origin of the cells can be derived from essentially any mammal, so long as the cells exhibit the characteristics that a7.low for secretion of the gene product of interest.
Cells to be used in a prosthetic chamber car. be adherent cell types, which can require isolation methods that temporarily digest or release the cells from their surrounding tissue. for example, smooth muscle cells can be obtained from a segment of veno~.~s or arterial tissue.
The smooth muscle cells can be obtained following enzymatic digestion in trypsin and co:llagenase and purified by positive selection using muscle cell-specific antigens such as von Willebrand factor (hejnieks et al., Bloc,_d, 92:1-7 (1998). Alternative7.y, cells can be isolated following digestion and further purified by centrifugation. The centrifugation can be performed in the presence of a gradient such as a sucrose gradient, WO 9916Z56Z PCT'NS99/12t16 which would allow for further separation of cell populations based on their density, Methods for the isolation of primary cells from a tissue source are well known in the art (see, for example, Freshney, Animal Cell ~~ltm re: A Practical Ap~oach, 2nd ed., IRL Press at Oxford University Press, New York (1992). Maintenance of the cells prior to modification and implantation can be as a cell suspension, adherent cell culture or as organ culture. Conditions for the maintenance and culture of l0 primary and clonal cells are well known in the art.
For therapeutic applications, the above cell types are additionally chosen to be implantable in an individual and remain viable in visrr~ without being substantially rejected by the host immune system.
15 Therefore, the donor origin of the cell type is a further characteristic that should be evaluated when selecting cells for therapeutic application. Those skilled in the art know what characteristics should be exhibited by cells to remain viable following implantation. Moreover, 20 methods well known in the art are available to augment the viability of cells following implantation into a recipient individual.
One characteristic for an implanted donor cell type is to exhibit substantial immunological 25 compatibility with the recipient individual. A cell is immunologically compatible if it is either histocompatible with recipient host antigens or if it exhibits sufficient similarity in cell surface antigens so as not to elicit an effective toast anti-graft immune 30 response. Specific examples of immunologically compatible cells include autologous cells isolated from the recipient individual and allogene:ic cells which have substantially matched major histocompatibility (MHC) or transplantation antigens with the recipient individual.
Immunological cornpatibility can be determined by antigen typing using methods well known in the art. Using such methods those skilled in the art will know or can determine what level of antigen similarity is necessary for a cell or cell population to be immunologically compatible with a recipient individual. Tolerable differences between a donor cell and a recipient can vary with different tissues and can be readily determined by those skilled in the art.
In addition to selecting cells which exhibit characteristics that maintain viability following implantation into a recipient individual, methods well known in the art can be used to reduce the severity of an anti-graft immune response. Such methods can be used to further increase the in vivo viability of immunologically compatible cells or to allow the in v~.vo viability of less than perfectly matched cells or of non-immunologically compatible cells. Therefore, for therapeutic applications, it is rlOt necessary to select a cell type from the recipient individual to achieve viability of the modified cell following implantation.
Instead, and as described further below, alternative methods can be employed which can be used in conjunction with essentially any donor cell t.o confer sufficient viability of the modified cells to achieve a particular therapeutic effect.
For example, in the case of partially matched or non-matched cells, immunosuppressive agents can be used to render the host immune system tolerable to engraftment of the implanted cells. The regiment and type of immunosuppressive agent to be administered will depend on the degree of MHC similarity between the modified donor cell and the recipient. Those skilled in the art know, or can determine, what level of histocompatibility between donor and recipient antigens is applicable for use with one or more immunosuppressive agents. Following standard clinical protocols, administration and dosing of such immunosuppressive agents can be adjusted to improve efficiency of engraftment and the viability of the cells of the invention. Specific examples of immunosuppressive agents useful for reducing a host anti-graft immune response include, for example. cyclosporin, corticosteroids, and the immunosuppressive antibody known in the art as OKT3.
Another method which can be used to confer sufficient viability on partially-matched or non-matched cells is through the masking of the cells or of one or more MHC antigens) to protect the cells from host immune surveillance. Such methods allow the use of nan-autologous cells in an individual. Methods for masking cells or MHC molecules are well known in the art and include, for example, physically protecting or concealing the cells, as we:l.l as disguising them, from host immune surveillance. Physically protecting t_he cells can be achieved, for example, by encapsulating the cells within a semi-permeable prosthetic chamber as. described previously. Such a barrier prevents contact of host immune cells such as T-cells with the cells contained within the chamber but. still allows secretion of the desired gene product into the ext.race:Llular space and circulation system. Alternatively, antigens can be disguised by treating them with binding molecules such as antibodies that mask surface antigens and prevent recognition by the immune system.

Immunologica:lly naive cells also can be used for constructing the impl.antable cells of the invention.
Immunologically naive cells are devoid of MHC antigens that are recognized by a host anti-graft immune response.
5 Alternatively, such cells can contain one or more antigens in a non-recognizable form or can contain modified antigens that faithfully mirror a broad spectrum of MHC antigens and are therefore recognized as self-antigens by most MHC molecules. The use of 10 immunologically naive cells therefore has the added advantage of circumventing the use of the above-described immunosuppressive methods for augmenting or conferring immunocompatibility onto partially or non-matched cells.
As with autologous or allogeneic cells, such 15 immunosuppressive methods can nevertheless be used in conjunction with immunologically naive cells to facilitate viability of the glucose-regulated insulin producing cells.
An immunologically naive cell, or broad 20 spectrum donor cell, can be obtained from a variety of undifferentiated tissue sources, as well as from immunologically privileged tissues. Undifferentiated tissue sources include:, for example, cells obtained from embryonic and fetal tissues. An additional source of 25 immunologically naive cells include stem cells and lineage-specific progenitor cells. These cells are capable of further differentiation to give i:ise to multiple different cell types. Stem cells can be obtained from embryonic, fetal and adult tissues using 30 methods well known to those skilled i.n the art. Such cells can be used directly or modified further to enhance their donor spectrum of activity.

wo 9sissssz prrrus~nzms Immunologically privileged tissue sources include those tissues which express, for example, alternative MHC antigens or immunosuppressive molecules.
A specific example of alternative MHC antigens are those expressed by placental cells which prevent maternal anti-fetal immune responses. Additionally, placental cells are also known to express local immuno-suppressive molecules which inhibit the activity of maternal immune cells.
An immunologically naive cell or other donor cell can be modified to express genes encoding, for example, alternative MHC or immuno-suppressive molecules which confer immune evasive characteristics. Such a broad spectrum donor cell, or similarly, any of the donor cells described previously, can be tested for immunological compatibility by determining its immunogenicity in the presence of recipient immune cells.
Methods for determining immunogenicity and criteria for compatibility are well known in the ax't and include, for example, a mixed lymphocyte reaction, a chromium release assay or a natural killer cell assay. Immunogenicity can be assessed by culturing donor cells together with lympohocyte effector cells obtained from a recipient individual and measuring the survival of the donor cell targets. The extent of survival of the donor cells is indicative of, and correlates with, the viability of the cells following implantation.
Once a cell type has been selected as described above, cells expressing a desired gene product in a secretable form are generated by introducing a vector expressing the encoding gene into the selected cell type.

An expressible nucleic acid encoding a desired gene product can be constructed using methods well known in the art. Such methods are described in, for example, Sambrook et al . (Molecul ar Cl,~n; ng: ~~~~a~t ,~,y Mai La1 , Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989)) and Ausubel et al. (~~;~rent Protocols in Molecular Bioloav, John Wiley & Sons, LVew York (1998)).
For example, a nucleic acid sequence encoding the gene product can be obtained using polymerase chain reaction (PCR) to amplify the sequence from an appropriate tissue or cell type. Encoding nucleic acids can be obtained by other methods known to those skilled in the art as well as by cloning or chemical synthesis using sequences available in scientific databases.
Once obtained the encoding gene is operationally linked t:o express.ion and regulatory elements which direct expression and ;>ecretion of the gene product into the extracellu.lar space. Such promoter and regulatory elements are also well know in the art and are selected for compatibility with the cell type to be implanted. For example, retrovir.al. promoter and enhancers achieve constitutively high levels of expression in a wide variety of different cell types, including smooth muscJ.sy cells. GJhereas a constitutive promoter yielding relatively strong expression levels in fibroblasts can be selected from a variety of extracellar matrix proteins such as fibronectin, collage and laminin, from structural genes such as actin, tubulin and intermediate filaments and from general housekeeping genes such as those in the glycolysis pathway. Selection of compatible express.i.<:>n and regulatory elements for a particular cell type a~.so is wel:L known to those skilled in the art.

WO 99/61562 PCT/US99/1a116 Promoters can be constitutive such as those described above, or alternatively they can be inducible.
A specific examples of :inducible promoters is the glucose regulated promoter describe below and in the Examples.
For example, glucose-regulated expression of a gene product of interest can be achieved using a variety of promoter elements that control expression of a downstream gene in response to changes in levels of glucose.
Elements that are induc.ible by glucose generally exhibit low activity in the absence of glucose and are up-regulated in the presence of. increased glucose or a metabolite within a glu~.:ose-associated pathway. For example, it is known that TGF-a promoter activity is responsive to glucose (~lcClain et al., Proc. Natl. Acad.
~,ci. USA 89:8150-8154 (1992): and Raja et al., Mol.
E~~dc~crinol. 5:514-420 (1991)). Also, sequences from other glucose-responsive promoter elements can be used such as promoter elements from wild-type insulin, fibroblast growth factor (FGF), epidermal growth factor (EGE), PC2 or PC3 genes (Sander et al., Proc. Natl. Acad_ Sci. USA 95:11572-11577 (1998)). Other glucose responsive promoter elements can be found, for example, in genes for acetyl-CoA carboxylase, 6-phosphofructo-2-kinase, and L-type pyruvate kinase (Zhang and Kim, Arch.
,~?ochem. Bionhvs. 15:227-232 (1997): Dupriez and Rousseau, D~,A Cell Ri_o1-. 16:1075-1085 (1997); Antoine et al., ~. Bioj. Chem. 272:17937-17943 (1997); and Kennedy et al . , ~,~o,l~ Chem.. 272 : 20636-2069U ( 1997 ) ) . A wide variety of other inducible promoters applicable for use in the methods of the invention are well known in the art. Selection of a particular inducible promoter will depend, for example, on the desired level of expression and as well as the choice o.f inducer to administer to a recipient individual.

WO 99/61,562 PCTIUS99/1Z116 Similarly, selection and operational linkage of the encoding nucleic acid to regulatory sequences for processing and secretian into the ext:racellular space is similarly well known in the art. E'or delivery of one or more desired gene products to the circulatory system it is necessary for the product to be secreted. Therefore, the encoding nucleic acids should contain a signal.
sequence for routing to the endoplasmic reticulum and secretion outside of the cell. The signal sequence can to be derived from the authentic gene for the product of interest, or alternatively from a heterologous gene and linked in frame for secretion into the extraceilular space. Signal sequences and methods of using them are well known in the art.
Vectors used in the methods of the invention can be any vector computable with the host cell or tissue where the desired gene product is to be expressed. For example, the vectors can be plasmi.d or viral based vectors. The vectors can be known, or constructed from components well known :in the art. Tn the case of a viral vector, the vector can contain, for example, at least one viral long terminal repeat and a promoter sequence upstream and operably linked to a nucleotide sequence encoding the gene product of interest, followed by at Z5 least one viral long terminal repeat and polyadenylation signal downstream of the sequence encoding the gene product of interest. Where multiple gene products are to be expressed, a single vector can be used which encodes more than one gene of interest, ear_h gene of interest can be expressed as separate transcription units.
Alternatively, multiple genes can be transcribed as a single transcription unit containing internal ribosome entry sites that allow expression of the genes from a polycistronic messenger RNA (Adam et al., J. Virol.

wo ~i6zssz Pcr~s~nzi~6 65:4985-4990 (1991)). Additional. nucleic acid sequences can be inserted into the vector using methods well known in the art.
Representative retroviral vectors suitable for 5 use in the invention and methods for their design are described for example, in Osborne and Miller, Proc. Natl.
8cad Sci. USA. 85:68'il-6855 (1988); Osborne et al., c. Natl. Acad. Sci. USA. 92:8055-8058 (1995); Ramesh et al., Nun. Acids. Rp,~~., ?.9:2697-a?700 (1996): and Hock 10 et al., lood. 74:876-881 (19891. Other vectors may also be used and are well known in the art, such as lentiviral vectors, DNA vectors, adenoviral vectors, pseudotype retroviral vectors, Epstein-Barr viral vectors, adeno-associated viral (AAV', vectors, vesicular stomatitis 15 virus-g (VSV-g), VL30 vectors, and liposome mediated vectors. AAV vectors provide an advantage in that they integrate into the host cell chromosome and do not substantially cause an immune response from the recipient host individual. Representative pl.asmid vectors include, 20 pcDNA and various other mammalian cwell expression vectors which are commercially available through a variety of sources.
Methods for introducing such vectors into a cell are also well known in the art. (see for example, 25 Osborne et al., supra (1995)). One method of introducing a vector into a cell :is by transfec:tion of plasmid or DNA
vectors. Transfection methods are well known in the art and include, for example, calcium phosphate precipitation, electroporation, liposome-mediated 30 transfection, and microinjection as described, for example, in Sambrook .at al. supra (1989) and Ausubel et al., supra (1998). Alternatively, a retroviral or an AAV
vector can be transduced into a cell. Methods for transduction of retroviral and adenoviral-type vectors are also well known in the art and are described further below in the Examples.
Following transfection or transduction of cells S with vectors of the invention, the cells are selected using a selectable marker that is either on the same vector as the gene of interest or is co-transfected on a separate vector. Methods of selecting cells for expression of a selectable marker' encoded by a transfected vector are well known to those skilled in the art (see, for example, Ausubel et al. supra (1998)).
Following selection, an isolated population of cells expressing the desired gene product or products is obtained.
Verification that the population of cells expresses and secretes the desired gene product or products can be determined using methods well known in the art. For example, a modified population of cells can be verified for the ability to secrete the gene product assaying the amount of product secreted into the culture media under expression-promoting conditions. The can be measured by, for example, enzyme-linked immunoaffinity assay, radioimmunoassay or by a functional assay for one of the known biological functions of t:he gene product.
Such assays are well known to those skilled in the art.
Additional methods of selecting cells expressing the desired gene product include Northern analysis and solution hybridization of mRNA obtained from the cells, in situ hybridization, immunohistology, and immunofluorescence using antibodies specific for the gene product. Further selection of a population of cells suitable for use in the invention can be performed using in vivo models known to those skilled in the art. Once a population of modified cells has been obtained, the cells can be seeded into a prosthetic chamber and implanted into a recipient individual.
The invention provides a method of systemic delivery of a gene product wherein the' gene praduct is selected from the group consisting of c°ytokines, hormones and growth factors. The secreted gene product can be Epo and insulin.
The methods of the invention are effective for expressing essentially any gene product which can have an effect on a target disease or pathological condition if it can, for example, effect a change in the phenotypic or biochemical characteristics of the target tissue's cellular constituents by direct contact of the secreted gene product with the target cells, or alternatively, by indirect contact with one or mare factors, including other gene products, induced by the secreted gene product with the target cells. Indirect effects of the secreted gene product also include, for example, negatively regulating a factor which plays a detrimental role in the target disease or pathological condition by inducing down regulation of that factor through secretion of a negative regulator using the methods of the invention. Therefore, the methods for treating a pathology mediated by a deficiency of a gene product of the invention are directed to those pathologies having deficiencies in the expression or regulation of a extracel:lular gene product, including gene products found systemically, or having their effect via the systemic system. Those skilled in the art will know which particular gene product wi.Ll have therapeutic or beneficia.L effects on a particular disease and are applicable for use in the methods of the invention given the teachings and guidance provided herein.
For example, the methods of the invention can be used to implant an effective amount of cells secreting a wide variety of genEy products which reduce the severity of a number of diseases or pathological conditions.
Exemplarily gene products include, for example, hormones, cytokines, growth factors, blood clotting factors and enzymes. A specific example of a clotting factor is factor IX which can bE:~ used for reducing the severity of individuals with hemo~>hilia. Glucocerebrosidase is a specific example of aru enzyme which can be secreted from modified cells and implanted to treat the severity of Gauchers disease. Other gene products and categories of gene products which can be systemically delivered using the modified cells as implantab.Lc vehic7_es are known to those skilled in the drt and similarly applicable for use in the methods so the invention.
Hormones, cytokines and growth factors are three classes of factors that carr be secreted by implanted cells into a recipient to achieve a wide range of beneficial effects, and thus, reduce the severity of the condition. For example, Epo is a hormone which stimulates red blood cell p.roduct.ion. Secretion of this cytokine from engrafted cells following implantation can be used in conjunctiorn with a variety of diseases and pathological conditions to augment production of red cells. Such diseases and pathological conditions include, for example, organ and tissuEe transplants, anemia, blood transfusion, renal failure and human immunodeficiency viru:~ infection.

WO 99/62562 PC1'NS99I12116 Another hormone that can be secreted from implanted cells to trE:~at or reduce the severity of a disease is insulin. ':n diabetic individuals blood glucose can be high due to the lack of insulin production. Secretiorn of this hormone from engrafted cells following implantation will facilitate cellular uptake of blood glucose following digestion of a food in a diabetic individual. Moreover, the insulin encoding gene can be placed under the control of a glucose regulated promoter to achieve regulation of insulin production that parallels increases in blood sugar and achieves glucose homes>stasis similar t=o that of normal individuals. Therefore, the long term engraftment of insulin producing cells will alleviate the need for intravenous injection of insulin.
Interleukins are a class of immune-related cytokines which can be used to treat a wide variety of diseases and pathological conditions. These molecules function, for example, in immune cell regulation and can be used to augment immune responses for the treatment of immune disorders, including autoimrnune diseases, as well as for the treatment c>t cancer. Far example, in immune related disorders, regulatory T cells can be stimulated with interleukins to augment their ability to induce or inhibit an immune response. Similarly, in cancer treatment, immune celi.s can be stinnulated with interleukins to facilitate immune responses against the pathological cells or to increase the effectiveness of other therapies.
Other cytokines which function similarly can also be used either by themselves ar in conjunction with an interleukin. ThosE:~ skilled in the art know the physiological and regi.ilatory effects of a cytokine and can therefore select the appropriate molecule or combination of molecules to effectively treat or reduce the severity of an immune disorder or cancer. Therefore, interleukins are described herein as exemplarily 5 cytokines for the treatment of these pathological conditions and can readily be substituted or combined with another cytokines known to those skilled in the art to have an effect on a target disease such as an immune-related disease or cancer. Other diseases and 10 pathological conditions exist which can similarly be treated through the implantation and engraftment of cells expressing a number of different cytokines. The treatment of these conditions by expressing cytokines having known therapeutic effects can similarly be 15 accomplished using the method of the invention.
Growth factars such as granulocyte-colony stimulating factor (G-CSF), granulocyte, macrophage-colony stimulating factor IGM-CSF), and related factors which stimulate cell proliferation and differentiation 20 within the hematopoietic cell lineages also can be used for treatment or reduction in severity of a variety of diseases and pathological conditions using the methods described herein. For example, the stimulation of leukocyte production following bone marrow 25 transplantations can be increased by implanting into the bone marrow recipient cells expressing G-CSF or GM'CSF.
These cells are necessary for immune surveillance and are critical for protection against pathogenic organisms and therefore early survival of a recipient individual 30 following bone marrow ablation and transplantation.
Other factors within the hematopoietic lineage similarly induce the differentiation and proliferation of other cell lineages and also can be secreted by implanted cells WO 99/62562 PC'TlUS99/12116 as described herein to stimulate some or all of the cell lineages of this system.
A variety of growth factors other than the specific examples listed above also can be used in the methods of the invention. Such growth factors include, for example, insulin-like growth factor (IGF), nerve growth factor (NGF), epidermal growth factor (EGF), inhibins, tumor growth :factor (TGF), tumor necrosis factor (TNF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), stem cell factor, leukemia inhibitory factor and thrombopoietin. Such factors and their biological effects as well as those of the intcrleukin family of cytokines can be found described in" for example, Molecular Bioloqy and BiotechnoloUV, Ed lt.A. Myers, VCH
Publishers, Inc., New 'York, N.Y., 1995, which is incorporated herein by reference. Other growth factors, hormones and cytokines known in the art; can similarly be used in the methods of the invention to treat or reduce the severity of a disease or pathological condition. The selection of a particu.iar factor or factors to be secreted using the cell implantation methods of the invention can be made c:liven the tea<~hings and guidance provided herein together with that which is known in the art for the treatment of a particular disease.
It is understood that modifications which do not substantially affec t the activity of the various embodiments of this invention are also included within the definition of the invention pro~.rided herein.
Accordingly, the following examples are intended to illustrate but not limit. the present: invention.

EXAMPLE I
Long-Term In Yivo Expression of Therapeutic Levels of Ervthropoietin This Example describes the use of a prosthetic chamber for systemic, long-term delivery of Epo from implanted cells to achieve elevated levels of red blood cell production.
Smooth muscle cells were used as the implantation and delivery vehicle for Epo secretion. The site of implantation was in the digestive tract between the tunics serosa and the tunics muscularis. Briefly, expression and secretion of Epo was accomplished from smooth muscle cells transduced with a retroviral vector expressing the rat Epo cDNA. The retr.oviral vector, LrEpSN, was made by inserting an EcoRl:-BamHI fragment of the rat Epo cDNA into vector LXSN (Usborne et al., 1995, supra; Miller et al., Biotechniques 7:980 (1989)) A
plasmid containing the rat erythropoietin gene was obtained from Drs. JPR. Boissel and HF Bunn, and is describe in Wen et a:L., Blood 82:1507 (1993). The control vector, LASH, was made by inserting human ADA
cDNA into LPNSN-2, a vector encoding human purine nucleoside phosphorylase, as described by Hock et al., Blood 74:876-881 (1989). Similarly, the amphot.ropic retroviruses were generated as described by Hock et al.
Rat smooth muscle cells were used for implantation following transduction with the above described vectors. Briefly, rat smooth muscle cell cultures were prepared ;by enzymatic digestion of the aorta from a male Fisher 394 rat, and the cells were characterized by positive staining for muscle cell~-specific actins with HHF35 antibody and negative staining for von Willebrand factor, which is arr endothelial cell specific marker (see f:or example, teary et al., Hum. Gene Ther. 5:1213 (1994)). Primary cultures of rat smooth muscle cells as well as ecotropic FESUl and amphotropic PA317 retrovirus packaging cell lines, and NIH 3T3 thymidine kinase negative cells were grown in Dulbecco/Vogt-modified Eagle's medium (DMEM) supplemented with 10$ fetal bovine serum in humidified 5°~ C0~ at 37°C
(Miller et al., 1989, supra, and Miller and Buttimore Mol. Cell Biol. 6:2895 (1986)). For studies showing the cell distribution in PTFE implants, LrEpSN transduced cells were first labeled with the fluorescent marker 1,1'dioctadecyl-3,3,3'3-'tetramethylindocarbocyanine perchlorate(DiI) as described by Clowes et al., ,1. Clin.
Invest. 93: 699 (1994).
To transduce the cells, early passage smooth muscle cells were exposed to 16-hr vii-us harvests from PA317-LrEpSN or PA317-LASN amphotropic virus-producing cell lines for a period of 29 hr in the presence of polybrene (4 ~.g/ml). Infected cells were selected ir;
medium containing 6418 at 1 mg/ml. Vascular smooth muscle cells infected with LrEpSN and selected in 1 mg/ml G-418 antibiotic secreted 5.7 milliunits/29 h per 10~ cells of Epo.
To achieve cell implantation, two PTFE rings per animal were positioned under the serosal. plane of the rat stomach to create an area above the muscle layer enclosed by the serosa membrane. Figure 1 shows a schematic cross sectiUn of a PTFE chamber implanted under the tunica serosa of the stomach. The cross section shows a view of a pocket which is created by a PTFE ring when positioned on the muscle layer and seeded with transduced vascular smooth muscle cells.
Briefly, rats were anesthetized by intraperitoneal(IP) injection with 94mg/kg ketamine, 5mg/kg xylazine, and 0.5mg/kg acepromazine. All rats received 0.04 mg dexamethasone IP just prior to surgery.
An area from the thoracic inlet to the pubis was prepared for surgery and a 3cm m:idline abdominal incision was made from the xyphoid to the umbilicus. The stomach was temporarily exteriorized and held in place with a mosquito hemostat. A 0.5 crn superficial incision was made in the capsule on the cranial face of the body of the stomach and a small pocket of approximately 0.6 cm diameter was created under the capsule using blunt dissection. A small PTFE ring (inner diameter 4mm, outer diameter 6 mm) was inserted into the pocket and sutured in place using 5-0 maxon on a taper needle in a simple continuous pattern. The suture material was drawn tightly to constrict the ring to a final inner diameter of 2 to 3mm before finishing the knot. The fibrous tunic directly overlying the :ring was cryofrozen using a steel probe, and the ring was mechanically elevated to prevent the freezing of the underlying muscular layer to minimize tissue damage.
Prior to cell implantation, the PTFE ring was rinsed with 0.9% saline to remove any blood clots formed during surgery. Cell alz.quots of 1 x lU' cells/50 ml media were then introduced into the center of the ring through a 29-g intravenous (IV) catheter. Animals received two rings each containing 1 x 10" transduced vascular smooth muscle cells expressing either Epo or human ADA.

Claims (36)

Claims

1. A method of systemic delivery of a gene product compri-sing implanting within the wall of the digestive tract of an individual a prosthetic chamber containing an effecti-ve amount of cells secreting said gene product.

2. The method of claim 1, further comprising implanting said prosthetic chamber within or proximal to a connective tissue layer.

3. The method of claim 3, further comprising implanting said prosthetic chamber between the tunics serosa and the tunics muscularis of said digestive tract wall.

4. The method of claim 1, wherein said prosthetic chamber comprises an outer diameter or width of the chamber being greater than about 10 mm, preferably between about 7.0 and 10 mm, more preferably between about 3 and 6 mm.

5. The method of claim 1, wherein said cells secreting said gene product are smooth muscle cells or fibroblasts.

6. The method of claim 1, wherein said effective amount of cells secreting said gene product comprises a population of about 1x10 5 cells or greater, preferably about 1x10 6 cells or greater, more preferably about 1x10 7 cells or greater.

7. The method of claim 1, wherein said gene product is se-lected from the group consisting of cytokines, hormones, growth factors and clotting factors.

8. The method of claim 7, wherein said gene product is Epo or insulin.

9. A method of systemic delivery of a gene product compri-sing implanting within the wall of the digestive tract of an individual a prosthetic chamber containing an effecti-ve amount of cells modified for inducible expression of seid gene product, and inducing expression of said gene product.

10. The method of claim 9, further comprising implanting said prosthetic chamber within or proximal to a connective tissue layer.

11. The method of claim 9, further comprising implanting said prosthetic chamber between the tunics serosa and the tunics muscularis of said digestive tract wall.

12. The method of claim 9, wherein said prosthetic chamber comprises an outer diameter or width of the chamber being greater than about 10 mm, preferably between about 7.0 and 10 mm, more preferably between about 3 and 6 mm.

13. The method of claim 9, wherein said cells secreting said gene product are smooth muscle cells or fibroblasts.

14. The method of claim 9, wherein said effective amount of cells secreting said gene product comprises a population of about 1x10 5 cells or greater, preferably about 1x10 6 cells or greater, more preferably about 1x10 7 cells or greater.

15. The method of claim 9, wherein said gene product is se-lected from the group consisting of cytokines, hormones, growth factors and clotting factors.

16. The method of claim 15, wherein said gene product is, Epo or insulin.

17. The method of claim 9, wherein said cells modified for inducible expression of said gene product comprise a nucleic acid encoding said gene product operable linked to an inducible promoter/regulatory element.

18. The method of claim 9, wherein said induction of said gene product expression comprises administering to said individual an effective amount of a compound which in-creases the activity of said promoter/regulatory element.

19. A method of treating a pathology mediated by the defi-ciency of a gene product comprising implanting within the wall of the digestive tract of an individual a prosthetic chamber containing an effective amount of cells expres-sing therapeutic levels of said deficient gene product.

20. The method of claim 19, further comprising implanting said prosthetic chamber within or proximal to a connec-tive tissue layer.

21. A method of claim 19, further comprising implanting said prosthetic chamber between the tunica serosa and the tunica muscularis of said digestive tract wall.

22. The method of claim 19, wherein said prosthetic chamber comprises an outer diameter or width of the chamber being greater than about 10 mm, preferably between about 7.0 and 10 mm, more preferably between about 3 and 6 mm.

23. The method of claim 19, wherein said cells secreting said gene product are smooth muscle cells or fibroblasts.

24. The method of claim 19, wherein said effective amount of cells secreting said gene product comprises a population of about 1x10 5 cells or greater, preferably about 1x106 cells or greater, more preferably about 1x10 6 cells or greater.

25. The method of claim 19, wherein said gene product is selected from the group consisting of cytokines, hormo-nes, growth factors and clotting factors.

26. The method of claim 25, wherein said gene product is Epo or insulin.

27. A method of treating a pathology mediated by the defi-ciency of a gene product comprising implanting within the wall of the digestive tract of an individual a prosthetic chamber containing an effective amout of cells modified for inducible expression of therapeutic levels of said gene product, and inducing expression of therapeutic levels of said gene product.

28. The method of claim 27, further comprising implanting said prosthetic chamber within ar proximal to a connecti-ve tissue layer.

29. The method of claim 27, further comprising implanting said prosthetic chamber between the tunica serosa and the tunica muscularis of said digestive tract wall.

30. The method of claim 27, wherein said prosthetic chamber comprises an outer diameter or width of the chamber being greater than about 10 mm, preferably between about 7.0 and 10 mm, more preferably between about 3 and 6 mm.

31. The method of claim 27, wherein said cells secreting said gene product are smooth muscle cells or fibroblasts.

32. The method of claim 27, wherein said effective amount of cells secreting said gene product comprises a population of about 1x10 5 cells or greater, preferably about 1x10 6 cells or greater, more preferably about 1x10 7 cells or greater.

33. The method of claim 27, wherein said gene product is selected from the group consisting of cytokines, hormo-nes, growth factors and clotting factors.

34. The method of claim 33, wherein said gene product is Epo or insulin.

35. The method of claim 27, wherein said cells modified for inducible expression of said gene product comprise a nucleic acid encoding said gene product operable linked to an inducible promoter/regulatory element.

36. The method of claim 27, wherein said induction of said gene product expression comprises administering to said individual an effective amount of a compound which in-creases the activity of said promoter/regulatory element.